Inhibition of monoacylglycerol lipase reduces nicotine reward in the conditioned place preference test in male mice.

Nicotine, the primary psychoactive component in tobacco, plays a major role in the initiation and maintenance of tobacco dependence and addiction, a leading cause of preventable death worldwide. An essential need thus exists for more effective pharmacotherapies for nicotine-use cessation. Previous reports suggest that pharmacological and genetic blockade of CB1 receptors attenuate nicotine reinforcement and reward; while exogenous agonists enhanced these abuse-related behaviors. In this study, we utilized complementary genetic and pharmacologic approaches to test the hypothesis that increasing the levels of the endocannabinoid 2-arachindonoylglycerol (2-AG), will enhance nicotine reward by stimulating neuronal CB1 receptors. Contrary to our hypothesis, we found that inhibition of monoacylglycerol lipase (MAGL), the primary catabolic enzyme of 2-AG, attenuates nicotine conditioned place preference (CPP) in mice, through a non-CB1 receptor-mediated mechanism. MAGL inhibition did not alter palatable food reward or Lithium Chloride (LiCl) aversion. In support of our findings, repeated MAGL inhibition did not induce a reduction in CB1 brain receptor levels or hinder function. To explore the potential mechanism of action, we investigated if MAGL inhibition affected other fatty acid levels in our CPP paradigm. Indeed, MAGL inhibition caused a concomitant decrease in arachidonic acid (AA) levels in various brain regions of interest, suggesting an AA cascade-dependent mechanism. This idea is supported by dose-dependent attenuation of nicotine preference by the selective COX-2 inhibitors valdecoxib and LM-4131. Collectively, these findings, along with our reported studies on nicotine withdrawal, suggest that inhibition of MAGL represents a promising new target for the development of pharmacotherapies to treat nicotine dependence.

A novel fluorophosphonate inhibitor of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol with potential anti-obesity effects.

BACKGROUND AND PURPOSE: The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via DAG lipases (DAGL) α and ß is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects. EXPERIMENTAL APPROACH: Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice. KEY RESULTS: Of the new compounds, O-7460 exhibited the highest potency (IC50 = 690 nM) against the human recombinant DAGLα, and selectivity (IC50 > 10 µM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations ≥ 10 µM, and showed that this compound has only one major 'off-target', that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB1 or CB2 cannabinoid receptors (Ki > 10 µM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 µM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0-12 mg·kg⁻¹, i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight. CONCLUSIONS AND IMPLICATIONS: O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions.

The monoacylglycerol lipase inhibitor JZL184 suppresses inflammatory pain in the mouse carrageenan model.

AIM: The present study tested whether the selective monoacylglycerol lipase (MAGL) inhibitor JZL184 would reduce allodynia and paw edema in the carrageenan test. MAIN METHODS: The anti-edematous and anti-allodynic effects of JZL184 were compared to those of PF-3845, an inhibitor of fatty acid amide hydrolase (FAAH), and diclofenac, a non-selective cyclooxygenase inhibitor. Cannabinoid receptor involvement in the anti-edematous and anti-allodynic effects of JZL184 was evaluated by administration of the respective CB1 and CB2 receptor antagonists rimonabant and SR144528 as well as with CB1(-/-) and CB2(-/-) mice. JZL184 (1.6, 4, 16, or 40mg/kg) was administered for six days to assess tolerance. KEY FINDINGS: JZL184 administered before or after carrageenan significantly attenuated carrageenan-induced paw edema and mechanical allodynia. Complementary genetic and pharmacological approaches revealed that the anti-allodynic effects of JZL184 required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-edematous actions. Importantly, both the anti-edematous and anti-allodynic effects underwent tolerance following repeated injections of high dose JZL184 (16 or 40mg/kg), but repeated administration of low dose JZL184 (4mg/kg) retained efficacy. SIGNIFICANCE: These results suggest that the MAGL inhibitor JZL184 reduces inflammatory nociception through the activation of both CB1 and CB2 receptors, with no evidence of tolerance following repeated administration of low doses.

Inhibition of monoacylglycerol lipase attenuates nonsteroidal anti-inflammatory drug-induced gastric hemorrhages in mice.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used analgesics, but can cause gastric and esophageal hemorrhages, erosion, and ulceration. The endogenous cannabinoid (endocannabinoid; eCB) system possesses several potential targets to reduce gastric inflammatory states, including cannabinoid receptor type 1 (CB(1)), cannabinoid receptor type 2 (CB(2)), and enzymes that regulate the eCB ligands 2-arachidonoylglycerol (2-AG) and N-arachidonoyl ethanolamine (anandamide; AEA). In the presented study, we tested whether 4-nitrophenyl 4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184), a selective inhibitor of the primary catabolic enzyme of 2-AG, monoacylglycerol lipase (MAGL), would protect against NSAID-induced gastric damage. Food-deprived mice administered the nonselective cyclooxygenase inhibitor diclofenac sodium displayed gastric hemorrhages and increases in proinflammatory cytokines. JZL184, the proton pump inhibitor omeprazole (positive control), or the primary constituent of marijuana, Δ(9)-tetrahydrocannabinol (THC), significantly prevented diclofenac-induced gastric hemorrhages. JZL184 also increased stomach levels of 2-AG, but had no effect on AEA, arachidonic acid, or the prostaglandins E(2) and D(2). MAGL inhibition fully blocked diclofenac-induced increases in gastric levels of proinflammatory cytokines interleukin (IL)-1ß, IL-6, tumor necrosis factor α, and granulocyte colony-stimulating factor, as well as IL-10. Pharmacological inhibition or genetic deletion of CB(1) or CB(2) revealed that the gastroprotective effects of JZL184 and THC were mediated via CB(1). The antihemorrhagic effects of JZL184 persisted with repeated administration, indicating a lack of tolerance. These data indicate that increasing 2-AG protects against gastric damage induced by NSAIDs, and its primary catabolic enzyme MAGL offers a promising target for the development of analgesic therapeutics possessing gastroprotective properties.

Novel, potent THC/anandamide (hybrid) analogs.

The structure-activity relationship (SAR) of the end pentyl chain in anandamide (AEA) has been established to be very similar to that of Delta(9)-tetrahydrocannabinol (Delta(9)-THC). In order to broaden our understanding of the structural similarities between AEA and THC, hybrid structures 1-3 were designed. In these hybrids the aromatic ring of THC-DMH was linked to the AEA moiety through an ether linkage with the oxygen of the phenol of THC. Hybrid 1 (O-2220) was found to have very high binding affinity to CB1 receptors (K(i)=8.5 nM), and it is interesting to note that the orientation of the side chain with respect to the oxygen in the phenol is the same as in THCs. To further explore the SAR in this series the terminal carbon of the side chain was modified by adding different substituents. Several such analogs were synthesized and tested for their CB1 and CB2 binding affinities and in vivo activity (tetrad tests). The details of the synthesis and the biological activity of these compounds are described.

Development of the first potent and specific inhibitors of endocannabinoid biosynthesis.

Enzymes for the biosynthesis and degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG) have been cloned and are the sn-1-selective-diacylglycerol lipases alpha and beta (DAGLalpha and beta) and the monoacylglycerol lipase (MAGL), respectively. Here, we used membranes from COS cells over-expressing recombinant human DAGLalpha to screen new synthetic substances as DAGLalpha inhibitors, and cytosolic fractions from wild-type COS cells to look for MAGL inhibitors. DAGLalpha and MAGL activities were assessed by using sn-1-[14C]-oleoyl-2-arachidonoyl-glycerol and 2-[3H]-arachidonoylglycerol as substrates, respectively. We screened known compounds as well as new phosphonate derivatives of oleic acid and fluoro-phosphinoyl esters of different length. Apart from the general lipase inhibitor tetrahydrolipstatin (orlistat) (IC50 approximately 60 nM), the most potent inhibitors of DAGLalpha were O-3640 [octadec-9-enoic acid-1-(fluoro-methyl-phosphoryloxymethyl)-propylester] (IC50 = 500 nM), and O-3841 [octadec-9-enoic acid 1-methoxymethyl-2-(fluoro-methyl-phosphinoyloxy)-ethyl ester] (IC50 = 160 nM). Apart from being almost inactive on MAGL, these two compounds showed high selectivity over rat liver triacylglycerol lipase, rat N-acylphosphatidyl-ethanolamine-selective phospholipase D (involved in anandamide biosynthesis), rat fatty acid amide hydrolase and human recombinant cannabinoid CB1 and CB2 receptors. Methylarachidonoyl-fluorophosphonate and the novel compound UP-101 [O-ethyl-O-p-nitro-phenyl oleylphosphonate] inhibited both DAGLalpha and MAGL with similar potencies (IC50 = 0.8-0.1 and 3.7-3.2 microM, respectively). Thus, we report the first potent and specific inhibitors of the biosynthesis of 2-AG that may be used as pharmacological tools to investigate the biological role of this endocannabinoid.

Inhibition of cytosolic phospholipase A2alpha: hit to lead optimization.

Compound 1 was previously reported to be a potent inhibitor of cPLA(2)alpha in both artificial monomeric substrate and cell-based assays. However, 1 was inactive in whole blood assays previously used to characterize cyclooxygenase and lipoxygenase inhibitors. The IC(50) of 1 increased dramatically with cell number or lipid/detergent concentration. In an attempt to insert an electrophilic ketone between the indole and benzoic acid moieties, we discovered that increasing the distance between the two moieties gave a compound with activity in the GLU (7-hydroxycoumarinyl-gamma-linolenate) micelle assay, which contains lipid and detergent. Extensive structure-activity relationship work around this lead identified a potent pharmacophore for cPLA(2)alpha inhibition. The IC(50)s between the GLU micelle and rat whole blood assays correlated highly. No correlation was found for other parameters, including lipophilicity or acidity of the required acid functionality. Compounds 25, 39, and 94 emerged as potent, selective inhibitors of cPLA(2)alpha and represent well-validated starting points for further optimization.